PROJECT SUMMARY/ABSTRACT Cells maintain homeostasis by responding to their environment through regulated transcriptional programs. For many genes that are rapidly induced, RNA polymerase II (Pol II) is pre-bound in a paused state. Sensing of the stimulus triggers the switch to productive Pol II elongation, thereby resulting in production of full-length mRNAs. Promoter-proximal pausing events are common, but are not observed at all inducible genes, including the metallothionein genes that are induced upon metal stress. As it is largely unclear how the rapid induction of the Drosophila metallothionein A gene (MtnA) is controlled, I performed a high-throughput RNAi screen and surprisingly found that the Integrator complex is a potent inhibitor of MtnA transcription. Integrator has a well- established role in 3' end processing of snRNAs, but I find that it is also directly recruited to the MtnA gene where it cleaves nascent MtnA transcripts to trigger transcription termination. The MtnA small RNAs are degraded by the RNA exosome, but can function as potent inhibitors of MtnA transcription when they accumulate. These data strongly suggest that Integrator cleavage provides an alternative mechanism to Pol II pausing as it helps keep full-length MtnA expression off in basal conditions, while also allowing Pol II to continuously engage the locus so that MtnA can be rapidly induced in response to metal stress. During the mentored phase, I will gain new training in biochemistry and high-throughput sequencing approaches to characterize the detailed mechanism by which Integrator-dependent premature termination events are triggered at MtnA and across the genome. In Aim 1, I will use reporter assays and immunoprecipitation experiments to determine how Integrator is recruited to the MtnA locus as well as how the MtnA small RNAs interfere with host gene induction. In Aim 2, I will use RNA-seq approaches to identify additional genes that are subjected to Integrator-dependent termination events. By examining sequence features at these regulated loci, this aim will reveal common features that dictate where/why Integrator cleaves to trigger transcription termination. In Aim 3, I will take full advantage of this training and determine in my own laboratory how the Integrator complex can have different functional effects at different gene loci. In particular, we will determine whether endonuclease activity is always required for Integrator function as well as address whether Integrator is modular and exists as sub-complexes that have different functional roles. In the short term, the proposed new training by my expert mentors will provide a strong foundation that I can continue to build upon in my own independent laboratory as we reveal how Integrator impacts inducible transcriptional events. The excellent training environment at UPenn will greatly facilitate the mentored research as well as endow me with the necessary skills to successfully transition to an independent faculty position.